Liver group 2 innate lymphoid cells regulate blood glucose levels through IL-13 signaling and suppression of gluconeogenesis

The liver stores glycogen and releases glucose into the blood upon increased energy demand. Group 2 innate lymphoid cells (ILC2) in adipose and pancreatic tissues are known for their involvement in glucose homeostasis, but the metabolic contribution of liver ILC2s has not been studied in detail. Here we show that liver ILC2s are directly involved in the regulation of blood glucose levels. Mechanistically, interleukin (IL)-33 treatment induces IL-13 production in liver ILC2s, while directly suppressing gluconeogenesis in a specific Hnf4a/G6pc-high primary hepatocyte cluster via Stat3. These hepatocytes significantly interact with liver ILC2s via IL-13/IL-13 receptor signaling. The results of transcriptional complex analysis and GATA3-ChIP-seq, ATAC-seq, and scRNA-seq trajectory analyses establish a positive regulatory role for the transcription factor GATA3 in IL-13 production by liver ILC2s, while AP-1 family members are shown to suppress IL-13 release. Thus, we identify a regulatory role and molecular mechanism by which liver ILC2s contribute to glucose homeostasis.

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Policy information about availability of computer code Data collection FACS CantoII(BD), FACSymphony A3 (BD)and FACSAriaIII(BD) were used for flow cytometry data collection. For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Portfolio guidelines for submitting code & software for further information.

March 2021
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy Bulk RNA-seq, ATAC-seq, ChIP-seq and single cell RNA-seq dataset will be deposited in the Gene Expression Omnibus (GEO) with the accession number GSE189287. The remaining data that support the findings of this study are available from the corresponding authors upon request. Source data are also provided as Source Data.

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All studies must disclose on these points even when the disclosure is negative.  (2018)). GraphPad PRISM software was used for statistical analyses. Unpaired t test were used to determine the statistical significance (p<0.05, one-tailed). We observed many statistically significant effects in the data, indicating that the effective sample size was sufficient for studying the phenomena of interest.
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The experimental findings were reliably reproduced with at least two independent experiments in Fig. 1a Fig. 5f is representative of 24 locations of 5 independent mice. Western blotting in Fig. 7f is representative figures of two independent experiments. We performed scRNA-seq of ILC2s using pooled tissues of 2-6 mice in each group (Fig. 3). ATAC-seq and ChIP-seq (Fig. 7c, d, Supplementary Fig. 6c, d, e) were performed with duplicates only except for GATA3-ChIP-seq from IL-33 treated lung ( Supplementary Fig. 6c right, 6e). RNA-seq (Fig. 7j) of cultured ILC2s were performed with triplicates.
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Although main investigator were not blinded to group allocation, all procedure of sampling and data collection (including blood glucose measuring, flowcytometry, RT-qPCR, ELISA, hepatocyte isolation) are performed not by single investigator, but some independent investigators. We also utilized many omics approach (Bulk-RNA-seq, ATAC-seq, ChIP-seq, single cell-RNAseq, MS/MS analysis, Metabolome) in this study, which enables us to interpret data without bias.

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Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research Laboratory animals BALB/c, nude (Foxnnu/nu), C57BL/6, NSG, NOD/Scid/Il2Rγnull (NSG), Il13-/-males of 6-8 weeks were used. All mice were allowed ad libitum access to food and water and were maintained in specific pathogen-free conditions in a 22°C temperature-controlled room with a 12 h light-12 h dark cycle. Humidity maintained in the range of 40-70%.

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The research proposals were reviewed by the ethics committee for animals at Chiba University.
Note that full information on the approval of the study protocol must also be provided in the manuscript. ChIP-seq data were mapped to the mouse genome build mm10 using Bowtie2 (v2.1.0) with default settings. ATAC-seq data were mapped to the mouse genome build mm10 using Bowtie2 (v2.1.0) with --broad option. For visualization in the IGV, HOMER makeTagDirectory and makeUCSCfile commands were used.